This invention relates to the application of genetic engineering techniques to plants. More specifically, the invention relates to a strategy for effecting changes in the composition of plant seed oils through the use of foreign DNA sequences which are derived from sources outside of the target plant gene pool.
Fatty acids are organic acids having a hydrocarbon chain of from about 4 to 24 carbons. Many different kinds of fatty acids are known which differ from each other in chain length, and in the presence, number and position of double bonds. In cells, fatty acids typically exist in covalently bound forms, the carboxyl portion being referred to as a fatty acyl group. The chain length and degree of saturation of these molecules is often depicted by the formula CX:Y, where xe2x80x9cXxe2x80x9d indicates number of carbons and xe2x80x9cYxe2x80x9d indicates number of double bonds. As the carbon chain of fatty acyl molecules always contains an even number of carbons, the formula xe2x80x9cC2Xxe2x80x9d may also be used to represent carbon chain length.
Fatty acyl groups are major components of many lipids, and their long, non-polar hydrocarbon chain is responsible for the water-insoluble nature of these lipid molecules. The type of covalent linkage of the fatty acyl group to other factors can vary. For example, in biosynthetic reactions they may be covalently bound via a thioester linkage to an acyl carrier protein (ACP) or to CoenzymeA (CoA), depending on the particular enzymatic reaction. In waxes, fatty acyl groups are linked to fatty alcohols via an ester linkage, and triacylglycerols have three fatty acyl groups linked to a glycerol molecule via an ester linkage.
The fatty acid composition of an oil determines its physical and chemical properties, and thus its uses. Plants, especially plant species which synthesize large amounts of oils in plant seeds, are an important source of oils both for edible and industrial uses.
The fatty acid composition of major oilseeds, ordered here by palmitate content, is shown in Table I. With the exception of laurate (C12:0) sources of coconut endosperm and palm kernel, the common edible oils all basically consist of 16:0, 18:0, 18:1 (oleate), 18:2 (linoleate), and 18:3 (linolenate).
Plant breeders have successfully modified the yield and fatty acid composition of various plant seed oils through programs of introducing desired traits by plant crosses and selection of progeny carrying the desired trait forward. Application of this technique thus is limited to traits which are found within the same plant species. Alternatively, exposure to mutagenic agents can also introduce traits which may produce changes in the composition of a plant seed oil. However, it is important to note that Fatty Acid Synthesis (FAS) occurs in leaf (chloroplasts) and seed tissue (proplastids). Thus, although a mutagenesis approach can sometimes result in a desired modification of the composition of a plant seed oil, it is difficult to effect a change which will not alter FAS in other tissues of the plant.
A wide range of novel vegetable oils compositions and/or improved means to obtain or manipulate fatty acid compositions, from biosynthetic or natural plant sources, are needed for a variety of intended uses. Plant breeding, even with mutagenesis, cannot meet this need and provide for the introduction of any oil traits which are outside of the target plant""s gene pool.
Various oils compositions are now in demand. For example, edible oil sources containing the minimum possible amounts of saturates, palmitate (C16:0) and stearate (C18:0) saturated fatty acids, are desired for dietary reasons and alternatives to current sources of highly saturated oil products, such as tropical oils, are also needed. Generating a spread of C4, C6 and C8 short chain 3-keto fatty acids could become a key improvement in polyhydroxybutyrate (PHB)-based biodegradable plastics made in bacteria and plants. Medium-chain fatty acids have special importance in the detergent and lubricant industries or in the formulation of edible oils with reduced caloric value or other health benefits. See for example, U.S. Pat. No. 4,863,753 and Barch, A. C. and Babayan, V. K., Am. J. Clin. Nat. (1982) 36:950-962. Longer chain fatty acids may have certain other utilities, i.e., C16 and C18 have particular uses in margarine and other solid oil-based products and very long chain fatty acids also have specialized uses, i.e., C22 is used to make peanut butter smoother. As such, a ready source of a variety of fatty acid lengths, including storage lipids which have incorporated differing chain length fatty acids in desired ratios, are desired for a variety of industrial and food use fields. Improved yield of current oilseed crops and the development of novel plant fatty acid compositions and oils products are also needed. Examples of novel plant fatty acid and oils products include fatty alcohols, epoxy fatty acids (e.g., biodegradable paint thinner), long chain liquid wax (e.g., jojoba oil substitute), hydroxylated fatty acids (motor lubricants) or cyclopropanated fatty acids (motor lubricants).
With the advent of genetic engineering, the ability to produce a transgenic plant containing any desired DNA sequence of interest is a reality. And with the development of basic plant biotechnology methodologies, many suggestions have been proposed for fatty acid modification. A good number of these strategies however, rely upon the insertion of genes isolated from organisms outside of the target plant species oftentimes traits from very divergent type plants to alter plant oils. It was not known whether such traits were limited to certain plant types. As one example, certain oil compositions appear to be limited to certain climates. Highly saturated oils, especially those high stearate (C18:0), are strongly correlated with tropical plant sources, e.g., oil palm, coconut. Temperate zone oilseeds are very typically highly unsaturated, e.g., corn, soybean, canola. Thus, the insertion of genes to achieve high stearate in a temperate crop would not meet the usual climatic condition for such trait.
Additionally, it was not known whether the introduced enzymes could effectively compete with the natural enzymes for substrate or whether it would be necessary to reduce the level of the endogenous enzymes to observe a modified fatty acid oil phenotype. Also, it was not known whether antisense technology could be used to influence the fatty acid pathway. In addition, it was not known, in the event that the composition of fatty acids were modified, whether the incorporation of such fatty acids into triglycerides would occur, whether transgenic seed with an altered oils composition would germinate, and to what extent if any, whether seed yield and/or oil yield from such seeds would be affected.
Moreover, in order to genetically engineer plants one must have in place the means to transfer genetic material to the plant in a stable and heritable manner. Additionally, one must have nucleic acid sequences capable of producing the desired phenotypic result, regulatory regions capable of directing the correct application of such sequences, and the like. Moreover, it should be appreciated that to produce a desired modified oils phenotype requires that the FAS pathway of the plant is modified to the extent that the ratios of reactants are modulated or changed.
Higher plants appear to synthesize fatty acids via a common metabolic pathway in plant plastid organelles (i.e., chloroplasts, proplastids, or other related organelles) as part of the FAS complex. (By fatty acid is meant free fatty acids and acyl-fatty acid groups.) Outside of plastid organelles, fatty acids are incorporated into triacylglycerols (triglycerides) and used in plant membranes and in neutral lipids. In developing seeds, where oils are produced and stored as sources of energy for future use, FAS occurs in proplastids.
The production of fatty acids begins in the plastid with the reaction between Acyl Carrier Protein (ACP) and acetyl-CoA to produce acetyl-ACP catalyzed by the enzyme, acetyl-CoA:ACP transacylase (ATA). Elongation of acetyl-ACP to 16- and 18-carbon fatty acids involves the cyclical action of the following sequence of reactions: condensation with a two-carbon unit from malonyl-ACP to form a xcex2-ketoacyl-ACP (xcex2-ketoacyl-ACP synthase), reduction of the keto-function to an alcohol (xcex2-ketoacyl-ACP reductase), dehydration to form an enoyl-ACP (xcex2-hydroxyacyl-ACP dehydrase), and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACP reductase). xcex2-ketoacyl-ACP synthase I, catalyzes elongation up to palmitoyl-ACP (C16:0), whereas xcex2-ketoacyl-ACP synthase II catalyzes the final elongation to stearoyl-ACP (C18:0). The longest chain fatty acids produced by the FAS are 18 carbons long. Monounsaturated fatty acids are also produced in the plastid through the action of a desaturase enzyme.
Common plant fatty acids, such as oleic, linoleic and xcex1-linolenic acids, are the result of sequential desaturation of stearate. The first desaturation step is the desaturation of stearoyl-ACP (C18:0) to form oleoyl-ACP (C18:1) in a reaction often catalyzed by a xcex94-9 desaturase, also often referred to as a xe2x80x9cstearoyl-ACP desaturasexe2x80x9d because of its high activity toward stearate the 18 carbon acyl-ACP. The desaturase enzyme functions to add a double bond at the ninth carbon in accordance with the following reaction (I):
Stearoyl-ACP+ferredoxin(II)+O2+2H+xe2x86x92oleoyl-ACP+ferredoxin(III)+2H2O.
In subsequent sequential steps for triglyceride production, polyunsaturated fatty acids may be produced. These desaturations occur outside of the plastid as a result of the action of membrane-bound enzymes. Difficulties in the solubilization of such membrane-bound enzymes has hindered efforts to characterize these enzymes. Additional double bonds are added at the twelve position carbon and thereafter, if added, at the 15 position carbon through the action of xcex94-12 desaturase and xcex94-15 desaturase, respectively. These xe2x80x9cdesaturasesxe2x80x9d thus create mono- or polyunsaturated fatty acids respectively.
A third xcex2-ketoacyl-ACP synthase has been reported in S. oleracea leaves having activity specific toward very short acyl-ACPs. This acetoacyl-ACP synthase or xe2x80x9cxcex2-ketoacyl-ACPxe2x80x9d synthase III has a preference to acetyl-CoA over acetyl-ACP, Jaworski, J. G., et al., Plant Phys. (1989) 90:41-44. It has been postulated that this enzyme may be an alternate pathway to begin FAS, instead of ATA.
The fatty acid composition of a plant cell is a reflection of the free fatty acid pool and the fatty acids (fatty acyl groups) incorporated into triglycerides. Thus, in a triglyceride molecule, represented as 
X, Y, and Z each represent fatty acids which may be the same or different from one another. Various combinations of fatty acids in the different positions in the triglyceride will alter the properties of triglyceride. For example, if the fatty acyl groups are mostly saturated fatty acids, then the triglyceride will be solid at room temperature. In general, however, vegetable oils tend to be mixtures of different triglycerides. The triglyceride oil properties are therefore a result of the combination of triglycerides which make up the oil, which are in turn influenced by their respective fatty acid compositions.
For example, cocoa-butter has certain desirable qualities (mouth feel, sharp melting point, etc.) which are a function of its triglyceride composition. Cocoa-butter contains approximately 24.4% palmitate (16:0), 34.5% stearate (18:0), 39.1% oleate (18:1) and 2% linoleate (18:2). Thus, in cocoa butter, palmitate-oleate-stearate (POS) (i.e., X, Y and Z, respectively, in Formula I) comprises almost 50% of triglyceride composition, with stearate-oleate-stearate (SOS) and palmitate-oleate-palmitate (POP) comprising the major portion of the balance at 39% and 16%, respectively, of the triglyceride composition. Other novel oils compositions of interest might include trierucin (three erucic) or a triglyceride with medium chain fatty acids in each position of the triglyceride molecule.
Thus, a variety of plant oils modifications are desired, including alternative crop sources for certain oils products and/or means to provide novel fatty acid compositions for plant seed.